In the progression of a carcinoma, transformed epithelial cells proliferate uncontrollably, eventually breaching through the basement membrane upon which they encounter the fibrillar ECM of the collagen-rich stroma. These cells then migrate through the stroma until they reach blood or lymphatic vessels and intravasate, thereby gaining access to other organ systems. The alignment of collagen fibrils within the tumor stroma is known to promote tumor cell invasion, allowing cells to migrate in a directionally persistent fashion towards neighboring vasculature. The small GTPases Rho and Rac are important players in interactions between the cell and its environment. Of note, Rac signaling is known to be a key mediator of directionally persistent cell migration. Although the specific population of cells responsible for fibrillar reorganization in the stroma is not known, it is likely that cell-generated force via Rho/ROCK driven contractility is a key player in this process. However, much of what is understood about the role of Rac and Rho in cell migration and force transduction has been derived from 2D flat surfaces which fail to recapitulate the 3D fibrillar microenvironment of the tumor stroma. Thus, the focus of the proposed work is to develop a synthetic fibrillar extracellular matrix to study the dynamic function of Rho/Rac signaling in a more physiological context. Importantly, this approach will allow for the independent control of structural features of the fibrillar microenvironment, which currently is not possible using collagen or fibrin gels. The following aims are proposed: Specific Aim 1: Characterize the effect of fibrillar matrix architecture on Rac activity and persistent cell migration. Cell morphology, adhesion, and migratory behavior on fibrillar networks varying in degrees of alignment (from completely isotropic to highly aligned) will be measured with time-lapse microscopy. Rac activity will be quantified and migration will be studied in the presence of Rac inhibitors or constitutively active mutants. Specific Aim 2: Determine if Rho-mediated fibrillar network reorganization is necessary for directional cell migration. Cell clusters will be placed on nonaligned (isotropic) fibrillar networks and fiber reorganization will be examined. Inhibitors of Rho and its downstream effectors will be introduced, as well as constitutively active mutants. Cell migration resulting from Rho-induced alignment will be quantified. Cell migration through fibrillar matrix is relevant to many disease settings, but given the special relevance to fibrosis and metastasis, we will use fibroblasts and melanoma cells as our experimental models for these studies. These investigations will rely heavily on biomaterial engineering, molecular biology, and live- cell imaging approaches in order to understand the interplay between the physical surroundings of the cell, intracellular signaling activities, and resulting cell migratory behavior. The proposed work will not only shed light on signaling mechanisms governing cell migration through extracellular matrix, but will also establish a new approach for devising matrices for the study of fundamental questions in cell biology. PUBLIC HEALTH RELEVANCE: Tumor cell metastasis, the movement of cancerous cells from the primary tumor to secondary sites throughout the body, is the predominant mechanism by which cancer kills. In order to reach blood and lymphatic vessels and gain access to other organ systems, these malignant cells must migrate through fibril- rich interstitial tissues. While tumor cell migration has been widely studied on glass surfaces, considerably less is known about the mechanisms which tumor cells use to migrate through fibrillar tissues. Therefore, the focus of the proposed work is to develop experimental approaches to study cell migration through fibrillar tissues, with the long term goal of identifying key signaling pathways that could become therapeutic targets that prevent metastasis and confine tumor cells to their primary site.